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Probe based on double-signal amplification triggered by target and application of probe

A dual-signal amplification and target technology, applied in the field of dual-signal amplification probes and their detection proteins, can solve the problems of long time, high cost, complicated operation, etc., achieve simple detection methods, reduce background noise, prevent The effect of false positive signal generation

Active Publication Date: 2015-07-22
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The second technical problem to be solved by the present invention is that the detection methods for proteins in the prior art are time-consuming, complicated to operate and high in cost, so as to provide a method for detecting protein molecules with simple operation and low cost

Method used

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  • Probe based on double-signal amplification triggered by target and application of probe
  • Probe based on double-signal amplification triggered by target and application of probe
  • Probe based on double-signal amplification triggered by target and application of probe

Examples

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Effect test

Embodiment 1

[0048] Example 1 Probe design based on target-triggered dual signal amplification

[0049] The probes for target-triggered dual signal amplification described in this example include: the first hairpin H1, the second hairpin H2 and the third hairpin H3, each of which has a sequence as shown in Table 1 below; The target protein is human thrombin.

[0050] Table 1 Probe sequences based on target-triggered dual signal amplification

[0051]

[0052] The first hairpin, the second hairpin and the third hairpin are all formed by the self-folding of the single-stranded linear molecule, and the complementary base pairing hybridization in the folded region, and the part of the double-stranded structure in the local region is the stem region, the part that does not form a double-strand structure and folds back is a ring region;

[0053] The first hairpin includes: an aptamer region I that can specifically recognize the target protein, and a first stem region II that hybridizes with...

Embodiment 2

[0068] Example 2 Detection of Target Protein Molecules at Different Concentrations Based on Target-Triggered Dual Signal Amplification Probes

[0069] The method for detecting target protein molecules based on target-triggered dual signal amplification probes of this embodiment includes the following steps:

[0070] First, take the solutions of the first hairpin, the second hairpin, and the third hairpin, heat them to 95° C., keep them for 5 minutes, then slowly lower them to room temperature, and set them aside. Take 10ml of dimethyl sulfoxide (DMSO) and 6.5mg of hemin to prepare 1mM hemin solution, and store it in a dark place at -20°C for future use. 4-Hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution was prepared for use, and the buffer solution included: 25 mM HEPES, 200 mM NaCl, 20 mM KCl, 1% DMSO, pH 7.4.

[0071] Prepare Tris-HCl buffer for use, the buffer includes: 20mM Tris-HCl, 200mM NaCl, 20mM KCl, 2mM MgCl 2 , pH value is 7.4. Take human α-throm...

Embodiment 3

[0075] Example 3 Signal Amplification Verification Test of Probes Based on Target-Triggered Double Signal Amplification

[0076] In this example, the following solutions were prepared according to the method in Example 2, and numbered a, b, c, d in sequence:

[0077] Sample a: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 200 nM;

[0078] Sample b: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 200 nM, and the concentration of the exonuclease III is 20 U;

[0079] Sample c: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 200 nM, and the concentration of the target thrombin is 1 nM;

[0080] Sample d: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 200 nM, the concentration of the exonucleas...

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Abstract

The invention provides a probe based on double-signal amplification triggered by a target and application of the probe. The probe comprises a first hairpin, a second hairpin and a third hairpin, wherein the first hairpin, the second hairpin and the third hairpin are formed by pairing hybridization of complementary bases in folding areas after single-chain linear molecules are folded back; the parts, forming double-chain structures in the partial areas, of the hairpins are stem areas, and the parts, not forming the double-chain structures and folded back, are ring-shaped areas. According to the probe provided by the invention, the self-assembly of hairpins is realized through target protein catalysis, signal amplification is achieved under the assistance of an excision enzyme III, obvious amplification of a detection signal is realized, and the detection sensitivity is greatly improved further; since the probe does not depend on template replication in the detection process, the problem that false positive occurs due to cross contamination in the detection process is avoided, generation of a false positive signal is effectively prevented and background noise is reduced greatly.

Description

technical field [0001] The invention belongs to the field of molecular bioinformatics, and in particular relates to a target-triggered double-signal amplification probe and a protein detection method and application thereof. Background technique [0002] The quantitative detection of protein plays a very important role in the field of biological research and drug diagnosis. However, since proteins are often at very low concentrations, highly sensitive analytical detection methods are often required to detect them. Therefore, it has always been the direction of people's research to detect protein molecules with high sensitivity, high selectivity, and simplicity. At present, enzyme-linked immunosorbent assay (ELISA) is the most commonly used method in the quantitative detection of proteins. Although this method can achieve the purpose of quantitative analysis, its detection sensitivity is still limited by the enzyme-substrate reaction. Trace protein molecules are detected, wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 吴昊邹霈诸飞帆刘娅灵王洪勇吴军
Owner JIANGSU INST OF NUCLEAR MEDICINE
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